The goal of this proposal is to engineer safer more efficacious vectors for use in human gene therapy. The development of safer and more efficient retroviral vectors has significant commercial potential in that these product vectors could become the standard for all retroviral gene therapy trials. Retroviral vectors have been utilized in clinical trials since1990, but poor efficacy remains a major problem. Not only are genes poorly expressed in many cell types, but the vectors are often silenced after a short period of time. Some tissue specificity can be obtained by using tissue-specific promoters, but traditional vectors do not allow genes to be expressed in a regulated manner nor in a development-specific manner. Furthermore, because retroviral vectors insert essentially randomly into transcriptionally active sites, insertional mutagenesis has always been a concern. With the occurrence of a leukemia-like disease in two French SCID-X1 patients who had been successfully treated with a murine retroviral vector, efforts to make retroviral vectors safer has taken on increased priority. We propose the following hypothesis: Retroviral vectors can be made safer and more efficacious by deleting viral cis-regulatory sequences and replacing them with human (or other eukaryotic) regulatory sequences. By removing the enhancer/promoter in the 3' LTR (i.e., using a SIN backbone), inserting a human locus control region together with its endogenous promoter, and providing insulator sequences, the potential for regulated gene expression would be greatly increased while the potential for activation of downstream oncogenes would be greatly reduced. The silencing of transduced genes would be minimized based on the speculation that mammalian cells may have a mechanism for recognizing viral regulatory sequences, and silencing them. Furthermore, the addition of a suicide gene translated from an IRES would add an additional safety feature. We propose to develop the design rules that will allow the construction of this new generation of retroviral and lentiviral vectors. These vectors would increase the efficacy and safety of gene therapy vectors to be used in clinical trials. [unreadable] [unreadable]